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Rapid detection of leaf spot pathogens on spinach using PCR and real-time PCR assays

Bo Liu: Univ of Arkansas

<div>Leaf spot diseases of spinach, caused by <em>Colletotrichum</em> spp., <em>Stemphylium botryosum,</em> <em>Cladosporium variabile,</em> and <em>Myrothecium verrucaria</em>, have become a major concern in spinach production in the U.S. Rapid detection and identification of these pathogens is necessary for epidemiological studies and effective disease management. Based on sequence data of the internal transcribed spacer (ITS) region of 30 representative isolates of each species (<em>Stemphylium botryosum,</em> <em>Cladosporium variabile,</em> and <em>Myrothecium verrucaria</em>), and the introns of glutamine synthetase (GS) and glyceraldehyde-3-phosphate dehydrogenase (GPDH) genes of 30 isolates of each species (<em>Colletotrichum</em> <em>dematium</em>, <em>C. coccodes</em>, and <em>C. truncatum</em>) from GenBank, and isolates of each pathogenic species recovered from the field, five sets of primers were designed and evaluated for their species-specificity. Results showed that two sets of primer pairs from each species were highly sensitive and specific for detection of each individual species using DNA recovered from single lesion on leaves by a rapid DNA extraction procedure. Moreover, a specific primer pair was designed and used for real-time PCR assay for rapid detection and quantification of each pathogen on spinach leaves. The PCR based diagnostic analysis can be completed within 24 h, compared with 1-2 weeks for doing isolations and identification. In addition, large-scale field testing was evaluated in order to further confirm the reliability of the newly developed molecular diagnostic techniques.</div>